Osteopenias are characterized by decreased mass density and calcification of bone. These diseases are generally attributable to inadequate osteoid synthesis thus causing an imbalance in the bone deposition to resorption ratio. Osteopenias include Paget's disease and primary and secondary osteoporosis. Various treatment protocols have been attempted the objects of which are to stimulate bone formation.
Growth hormone (GH) has been found to stimulate bone formation through stimulation of insulin-like growth factor-1 (IGF-1) production primarily by the liver and secondarily by osteoblasts. Osteoblasts are responsible for synthesis of bone matrix and are known to express osteoblasts are known to express GH receptors. IGF-1 further stimulates osteoblast proliferation. The biological half-lives of IGFs are increased when associated with IGF binding proteins that are also locally produced in the bone. It has been suggested that GH stimulates bone formation through a localized mechanism that increases IGF and IGF binding protein production which in turn stimulates osteoblast proliferation.
Hormonal treatments such as direct infusion of human growth hormone (hGH) have been attempted to stimulate bone deposition. They have not been successful to date. Therapies involving direct GH injections can produce adverse systemic effects. These include sterility or at least reduced fertility and adverse lactogenic effects. Because GH injections induce systemic stimulation of IGFs, which bind insulin receptors when present in excess, severe hypoglycemia may also result.
Attempts have been made to stimulate osteoblasts through localized production of GH in the bone so as to avoid adverse systemic effects through injection of GH. One approach achieved expression of a GH transgene in osteoblasts which stimulated increased growth in the long bones of transgenic mice. (Baker, A. R. et al. (1992) Mol. Cell. Biol 12: 5541-47). However, this approach would be difficult to adapt for treatment of osteopenias. After birth, osteoblast isolation from a human patient is highly impractical, thus eliminating use of transfected osteoblasts in any gene therapy. Further, no specific inducible regulatory sequences were included in the transgenic model to provide higher levels of expression, if desired in a given therapy.
Prior to the development of the present invention, no erythroid-specific gene therapy existed for localized and controlled stimulation of GH production to enhance bone deposition using a gene construct which could be up-regulatable based on therapeutic need and capable of being applied to more readily available and collectable tissues, such as bone marrow stem cells.
Erythroid-specific gene expression in transgenic non-human mammals has been previously reported in the art, principally using human beta globin genes. For example, WO 9(1/05041 published Apr. 18, 1991 discloses expression of human beta globin gene in erythroid tissue of transgenic non-human mammals having such expression enhanced through use of selected DNase hypersensitive sites (HS) from the locus control region (LCR) of the human beta globin gene. Palmiter, et al. Science 222, 809 (1983) discloses transgenic mice containing the human growth hormone gene fused to a metallothionein promoter sequence. These references neither suggest nor disclose expression of an erythroid-specific gene capable of being up-regulatable in response to therapeutic needs.
A juvenile beta globin gene (.beta..sup.c), possessed by artiodactyls (goats and sheep of the haplotype A), exhibits a unique developmental expression pattern. Most mammals have embryonic beta globin genes active early in development. These embryonic genes are then down-regulated in favor of beta globin genes encoding for adult forms of beta globin. Sheep of the A haplotype have a gene switching pattern comprising "embryonic-fetal-juvenile+adult-adult." When mature sheep become anemic or exposed to high altitude, which causes erythropoietin (EPO) levels to rise, the switching pattern can be reversed and .beta..sup.c up-regulated with adult beta globin being down regulated. The juvenile beta globin gene can also be reinduced artificially in healthy adult sheep through administration of EPO.
No gene construct known in the prior art is up-regulatable, erythroid-specific and ligatable to hGH gene for gene therapeutic treatment of osteopenias.